Probiotics and Human Health

By: Chloe McClanahan, BioFiles Edition 6.2

At the beginning of the 20th century, probiotics were thought to have a beneficial effect to the host by improving the intestinal microbial balance. Through his studies and observations, the Russian biologist Metchnikoff noted people from cultures which consumed large amounts of fermented milk, generally had higher lifespans. He proposed that lactic-acid bacteria create a more acidic environment as a result of fermentation, thereby suppressing the growth of proteolytic bacteria. Metchnikoff propagated the consumption of sour milk fermented with the Lactobacillus bulgaricus as a means of promoting intestinal balance and overall health.

Today, diverse studies report the benefits of probiotics, such as inhibitory effects on pathogens, aid in the management or prevention of chronic intestinal inflammatory diseases or atopic syndromes, and support to the immune system. Potential beneficial applications abound, researchers continue to evaluate the effictiveness and clarify the mechanisms of action of probiotics. Nevertheless, the use of probiotics has surged due to direct to consumer marketing and lenient regulatory control.

The efficacy of probiotics in shortening the duration of acute infectious diarrhea has been repeatedly confirmed but controlled studies need to be performed to guide the dosage, duration, and strain combination for specific patient groups. Similarly, the use of probiotics in gastrointestinal inflammatory diseases, including ulcerative colitis, Crohn's disease, and irritable bowel syndrome has been investigated but only a few randomized controlled trials have been conducted.

Current research is addressing the modulation of gastrointestinal immune function by probiotics. Evidence from animal model studies shows probiotic treatments provide protection from inflammation of the colon by down regulating inflammatory cytokines or inducing regulatory mechanisms. The examination of probiotic influence on dendritic cell function and cytokine secretion is ongoing. Recently, scientists in Finland have discovered cheese can help preserve and enhance the immune system of the elderly by acting as a carrier for probiotic bacteria. The research showed daily consumption of probiotic cheese helps to bolster immunity in elderly people. Cheese was confirmed to be an effective carrier for probiotics.

The cytoprotective potential of probiotics is also being investigated. A study reported exopolysaccharides released into the surrounding environment by the probiotic strain Bacillus coagulans RK-02 had significant antioxidant and free radical scavenging activity. Another study effectively used a probiotic formulation against acetaminophen induced hepatotoxicity.

Sigma Life Science provides the quality, service, and product selection to support the movement towards a deeper understanding of the relationship between probiotics, intestinal microbiota, and human health.

Bifidobacterium

Bifidobacterium is one of the most important probiotic bacteria used in the dairy industry. They are Gram-positive, non-motile, rodshaped, and often branched anaerobic bacteria. They were first isolated from a breast-fed infant by Henry Tissier of the Pasteur Institute. At that time, Tissier named the organism Bacillus bifidus communis . Bifidobacteria have a positive effect on the immune system and help to control intestinal pH. In addition, Bifidobacteria produce bacteriocins and bacteriocin-like inhibitory compounds which inhibit the growth of other bacteria.

Bifidobacteria posess many glycosylases able to degrade various plant or milk derived oligosaccharides. Several such enzymes were identified on the Bifidobacterium genome. The genomic sequencing of Bifidobacterium animalis subsp. lactis AD011 revealed a glycosyl hydrolase cluster containing a transcriptional regulator and an ABC transporter, as well as a fos gene cluster that is involved in the processing of prebiotics. Bifidobacteria are able to utilize a broad range of substrates as energy sources, such as plant polymers, glycoproteins, and glycoconjugates, as well as having specialized proteins for the catabolism of oligosaccharides.

Bifidobacteria also have a unique hexose metabolism called the bifid shunt. The key enzyme, fructose-6-phosphate phosphoketolase is not found in any other Gram-positive intestinal bacteria and therefore, provides an ideal target for a diagnostic test.

It was found that live Bifidobacterium lactis can directly counteract the harmful effects of coeliac-toxic gliadin and this may prove to be a future potential treatment of coeliac disease.

In adult intestines, only 3–6% of the fecal flora is composed of bifidobacteria, while in breast-fed infants bifidobacteria can constitute up to 90%. With increasing age, the number of bifidobacteria decreases. It was observed that babies and adults with lower numbers of bifidobacteria have a higher risk for diarrhea and allergies. For this reason, bifidobacteria are added as a probiotic supplement to infant formulas, drinks, yogurts, and a range of other products.

Because of the wide use of bifidobacteria, Sigma-Aldrich has developed a bifidobacteria selective medium (BSM), available as an agar or a broth, as a standard for quality control. This medium allows for fast and easy quality control of yogurt made with bifidobacteria and can be used to control the count of bifidobacteria. Bifidobacterium grow very well on this medium, while Lactobacillus and Streptococcus strains are inhibited. Bifidobacterium colonies grow within 24–48 hours (occasionally up to three days because of the highly selective conditions). The Bifidobacterium colonies are purple-brown, and therefore, are easy to differentiate from other organisms. In a Swiss governmental evaluation study for the enumeration of bifidobacteria in sour milk products, the traditional method was compared to Wilkins-Chalgren Agar with 100 mg/L mupirocine and BSM Agar. The traditional method produced statistically significant differences, while Wilkins-Chalgren Agar and BSM Agar showed similar results without any significant variances.

Lactobacillus

Lactobacilli are rod-shaped, Gram-positive, fermentative, facultative anaerobic or microaerophilic organotrophs. Normally they form straight rods, but under certain conditions spiral or coccobacillary forms have been observed. In most cases, they form chains of varying length. Lactobacilli belong to the lactic acid bacteria and comprise the major part of this group. As their name implies, they produce lactic acid and derive energy from the fermentation of lactose, glucose, and other sugars to lactate via homofermentative metabolism. About 85–90% of the sugar utilized in the fermentative process is converted to lactic acid. This acid producing mechanism inhibits growth of other organisms and favors the growth of lactobacilli that thrive in low pH environments. ATP is generated during the process by non-oxidative substrate-level phosphorylation.

Some lactobacilli strains were shown to produce, like bifidobacteria, a bacteriocinlike substance and are able to inhibit a broad range of pathogens. Lactobacilli also produce adhesins (proteins), which perform a vital role in recognizing specific host components (extracellular matrix) important for the bacterial adhesion and colonization at host surfaces, as well as in bacterial interaction with physiological and immunological processes.

In the last several years, several new Lactobacillus species have been introduced as probiotics, including Lactobacillus rhamnosus , Lactobacillus casei , and Lactobacillus johnsonii . As of this time, probiotics have not been used in the pharmaceutical industry due to the many open questions that remain to be answered.

Since lactobacilli prefer acidic conditions, natural extracts and juices from tomatoes and oranges, as well as other single metabolic acids (e.g. malic acid), are often used as media ingredients. Casein and yeast extract provide rich amino acid sources, and the maltose is used as a carbohydrate source for lactobacilli, which cannot utilize glucose as fermentable sugar. Fructose is the carbohydrate source of Lactobacillus fructivorans . Polysorbate, sorbitan monooleate and other related compounds act as a source of fatty acids and stimulate the lactic acid bacteria. Today, it is standard practice to differentiate lactobacilli based on their phenotype using selective media. Classical phenotypic tests for identification of lactobacilli are based on physiological characteristics, like motility, growth temperature, respiratory type, and growth in sodium chloride, as well as on diverse biochemical characteristics, such as fermentation type, metabolism of carbohydrate substrates, production of lactic acid isomers, coagulation of milk, and presence of specific enzymes like arginine dihydrolase. In Bergey’s Manual, Lactobacillus is described as a Gram-positive rod, nonspore forming, acid fast negative and catalase negative. The colony morphology on certain media is taken for the presumptive identification.

For the isolation, cultivation and enumeration of acid-tolerant spoilage microorganisms in fruit juice and fruit juice concentrates, in particular from citrus fruit, according to Hays, Troy, and Beisel. The prepared agar may be turbid after autoclaving due to orange extract.

HybriScan™ Detection

Molecular biology-based methods, like PCR, can be used for lactobacilli detection. However, they are often quite expensive. Sigma-Aldrich provides a revolutionary molecular biology method that is rapid, easy, and cost effective. Based on the detection of rRNA, this method completely avoids the need for PCR amplification. The sandwich hybridization test, called HybriScan, is performed on a microtiter plate. The range of lactobacilli detected by HybriScan tests is listed in the table. For more information about the test and the technical principles, please visit sigma-aldrich.com/hybriscan.

Fermentation: Enzymes, Substrates, and SCFA

The influence of probiotics on the metabolic activity of intestinal microbiota may be evaulated by detecting bacterial enzymatic activity in fecal samples, specifically β-glucosidase, β-glucuronidase, and urease activity. Experimental studies using probiotics have indicated a significant decrease in β-glucuronidase activity. A decrease in β-glucuronidase decreases the release of carcinogens from carcinogen-glucuronide conjugates within the colonic lumen.

Short-Chain Fatty Acids (SCFA)

Probiotics and prebiotics enhance SCFA end product synthesis. An increase in SCFA production lowers the gastrointestinal pH thus improving pathogen resistance and stimulating epithelial cell proliferation. The predominant SCFA, butyrate, is the preferred substrate for colonocytes which likely contributes to a normal colonocyte population and a lower cancer risk. SCFA production can be measured with gas chromatography analysis on fecal samples or intestinal modeling systems. However, due to normal colon absorption of 95% of the SCFA metabolites, stable isotope technology is a superior method of analysis.

Bile Salts and Bile Salt Hydrolase

Several Lactobacillus and Bifidobacterium probiotic strains exhibit bile salt hydrolase (BSH) activity and are suspected to play a role in combating the negative effects of bile via detoxification of bile salts. However, enhanced BSH activity is potentially detrimental to the host. More research is needed to understand this dynamic between probiotics and bile salts.